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The Regulation of Microecological Conditions on Tissue Culture of Tripterygium Wilfordii Hook.F

Author: LiJianJuan
Tutor: WuChengZuo; HongWei
School: Fujian Agriculture and Forestry University
Course: Ecology
Keywords: Tripterygium wilfordii Hook.F Tissue Culture Orthogonal Experimental Design Microecology Hormone
CLC: S567.19
Type: Master's thesis
Year: 2009
Downloads: 14
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Abstract


Tripterygium wilfordii Hook.F is a traditional Chinese herbal medicine with clearing away heat and toxic, Qufeng Tongluo, Shujin network of blood, relieving pain, stopping bleeding, and such as insecticidal efficacy. It has a good effects on rheuatiod arthritis, varieties of nephritis, refractory nephrotic syndrome, and lupus erythematosus and more than 10 types of other refractory diseases. Increasing demand for Tripterygium in recent years, the wild tripterygium resources is declining sharply. In order to integrate sustainable using of tripterygium resources and meeting the needs of production and linving of people, it is a great of importance and application prospects through using tissue culture method to cultivaterenewable tripterygium Miller. On the basis of tripterygium breeding, with the superior clones of materials, by the orthogonal experimental design method, superior clones of tripterygium rapid propagation of tissee culture conditions were optimized in this study. It can provide a theoretical basis and application foundation on seedling up the production factory tripterygium. The main conculutions were drawn as follows:1. The best explant was disease-free green leaves; the best way of sterilization was 70% alcohol(30s)+ 10% NaClO(15min) . The contaminated rate was lowest which was 21.08%, and the survive rate was highest which was 65.66%.2. The impact of basic medium type and concentration of hormone combinations were significant on callus tissue induction of Tripterygiumi. The calluse induction rate on MS was the highest. By the L16(45) orthogonal experiment design on hormone optimization, the impact of factors on callus induction of Tripterygium was followed by 2,4-D>NAA>KT, the best medium was MS+2,4-D1.5mg/L+ NAA1.5mg/L+KT0.1mg/L. The induction rate was 92.93%. Aand the best time of culture was for 40ds. The linear model of relationship between hormone and callus induction rate of Tripterygium was: y = 46.85 + 5.35 x1 + 5.79 x 2 ? 2.18x3( R = 0.681)3. Through L9(34) orthogonal experimental design it found that the impact of factors on callus proliferation of Tripterygium was followed by IAA>KTA>2,4-D. The optimum medium was MS+2,4-D1.0mg/L+IAA1.0mg/L+KT0.1mg/L+AgNO350mg/L.The relative growth rate was 485.08%. The best light conditions was in dark for a week , and then in light with 16h/d. The best time of culture was for 30ds. The linear model of relationship between hormone and relative growth rate of callus proliferation of Tripterygium was: y = ?8 3.42 + 44.12 x1 + 111.10 x 2 + 37.81x3( R = 0.654)4. The impact of basic medium type, hormone type and concentration of hormone combinations were significant on adventitious buds induction of Tripterygium. Adventitious buds induction of tripterygium need the medium with complete elements, high potassium, ammonium and nitrate content. Both 1/2MS and MS could induced the differentiation of adventitious buds, and the induction rate on MS was the highest; the adventitious buds induction of tripterygium needed interaction between KT and 6-BA; additionnally, only NAA most conducive to adventitious buds induction of tripterygium in auxinses. The result of L9(34) orthogonal experimental design showed that the impact of factors on adventitious buds induction of Tripterygium was followed by 6-BA>KT>NAA. The optimum medium was MS+NAA0.2mg/L+ KT0.5mg/L+ 6-BA1.5mg/L. The induction of adventitious buds was 73.68%. The best time of culture was for 60ds. The linear model of relationship between hormone and induction rate of adventitious buds of Tripterygium was: y = 93.79 ? 1.20 x1 ? 2.11x 2 ? 27.03x3( R = 0.894)5. The impact of concentration of hormone combinations was very significantly on adventitious buds proliferation of Tripterygium. By L9(34) orthogonal experimental design , it found that the impact of factors on adventitious buds proliferation of Tripterygium was followed by NAA>KT>6-BA.. The optimum medium was MS+NAA0.1mg/L+KT0.1mg/L+6-BA1.0mg/L, the proliferation multiple of adventitious buds was 3.56. The best time of culture was for 60ds. In order to maintain a certain of growth activity and proliferation coefficient, the height of bud seedling for proliferation must be over 2cm, and the subculture times was confined within 6 generations. The linear model of relationship between hormone and proliferation multiple of adventitious buds of Tripterygium was: y = 3.98 ? 0.38 x1 ? 0.32 x 2 ? 0.15x3( R = 0.599)6. By L9(34) orthogonal experimental design , it found that the impact of basic medium type and concentration of hormone combinations were not significant on rooting experiment of Tripterygium. The impact extent of factors on rooting rate of Tripterygium was followed by NAA>basic medium>KT; the average root length was follwed by basic medium>NAA>KT; the average root number was followed by NAA>basic medium>KT. The optimum medium was 1/2MS+NAA2.0mg/L+KT0.1mg/L+AC 0.50g/L. The rooting rate could be 100% with 2.77cm lengthe and the average root number was over 11.42 after 45ds. In order to maintain a certain of growth activity a the highth of bud seedling for rooting must be over 2~3cm. Tthe best time of culture was for 60ds. The model which could reflect the relationship betweem rooting rate of Tripterygium and different culure time was: 2y = 3.42 x1 + 0.03 x2? 18.63( R = 0.995) The model which could reflect the relationship betweem average root length of Tripterygium and different culure time was: y = 0.07 x1? 0.31( R = 0.995) The model which could reflect the relationship betweem average root number of Tripterygium and different culure time was: 2y = 0.37 x1 + 0.01x 2? 2.53( R = 0.991)7. The results showed that the length of tube seedling with height more than 4cm in height, root number three above and root length more than 2.0cm conducive to the survival on transplanting mostly, the optimum transplanting media was Perlite: vermiculite = 1:2, and the survival rate was 87% arfter 30ds.. Because the indexes of all have reached the reguirement of rapid propagation, the technical measures could meet needs of production.

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